Ion ampliseq targeted

Total RNAs were extracted, purified, and eluted from Formalin-Fixed Paraffin-Embedded (FFPE) tissue sections by the truXTRAC™ FFPE RNA Kit (Covaris, Inc., Woburn, MA) and then quantified using the Quant-iT RNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA). RNA-Seq libraries were generated using the Ion AmpliSeq™ targeted sequencing system (Thermo Fisher Scientific, Waltham, MA). A total of 395 genes were quantified, including 10 housekeeping (HK) genes as controls. After reverse transcription into complementary DNA (cDNA), the barcode adapter was ligated to the partially digested amplicon. Equimolar libraries were pooled after purification and normalization and subjected to the Ion Chef™ system (Thermo Fisher Scientific) for enrichment and template preparation. Afterwards, 200 bp sequencing was performed to obtain mapped reads (approximately 2-3 million per sample) using the Ion Torrent S5 system (Thermo Fisher Scientific). Next-generation sequencing (NGS) data were subsequently analyzed using Ion Torrent Suite software version 5.2.0 (Thermo Fisher Scientific) for NGS read alignment, reference mapping, variant calling, and data management. NGS read quality control and quality assurance were conducted using standardized criteria [15 (link)]. Reads of HK genes were used for gene expression normalization. Finally, normalized reads per million (nRPM) were log2-transformed.